Analytical and Forensic Toxicology

Analytical and Forensic 

Evan S. Schwarz, MD 

Washington University 

St. Louis, MO 

Barnes Jewish Hospital 

Toxicology

• Howard Greller 

• Jeffrey Brent 

• Adhi Sharma 

• Chuck McCay 

• Kent Olson 

Special Thanks! 

It was the break they had been waiting 

for; prints left at the crime scene

Content 

• Core Content of Medical Toxicology 

• Part 5: Analytical and Forensic Toxicology 

• LLSA Articles 

• Post-mortem Toxicology: What the dead 

can and cannot tell us. Clin Tox 

2003;41(1):47-56. 

• Is this urine really negative? J of Sub Abus 

Treat 207;33:33-42.

Laboratories 

• Clinical Laboratory Improvement 

Amendments of 1988 (CLIA) 

• Medical Lab Testing governed by federal 

regulations since 1992 

• Regulations apply to all lab testing of human 

specimens for medical purposes 

• All tests require the possession of an 

appropriate certificate

• Waived 

• Moderate complexity 

• High complexity 

Testing 

Moderate 

Record Keeping 

Written Procedures 

Laboratory Director 

Competency Testing 

Proficiency Testing 

Controls 

Inspection 

High 

Qualified onset supervisor 

Daily review of all results

Magic Answer Box? 

• Low volume, often old methodologies 

• Limits to every technique 

• Implies confirmation or exclusion 

• More toxic xenobiotics than 

named diseases

Magic Answer Box? 

• Old methodologies, limited samples 

• Limits to every technique 

• More toxic xenobiotics than 

named diseases

• Screening 

• Confirmation 

• Separation 

• Detection 

• Quantification 

Basics of analysis

Testing 

“You’re fired, Jack. The lab results 

just came back, and you tested positive for Coke.”

Assay Methods 

• Spot & spectrochemical 

• Immunoassays 

• Non-competitive 

• Competitive 

• Enzyme-multiplied immunoassay (EMIT) 

• Magnetic microparticle chemiluminescent 

competitive immunosassay 

• 

Microparticle capture immunoassay

Assay Methods 

• Chromatography 

• Thin layer liquid (TLC) 

• High performance (pressure) liquid 

(HPLC) 

• Gas chromatography (GC) 

• Mass spectroscopy (MS) 

• Inductively coupled plasma mass 

spectroscopy

• Simple 

• Rapid Reaction 

• Color change 

• Ferric chloride for 

salicylate 

• Unreliable (FP & FN) 

• Visual 

interpretation 

Spot tests

• Salicylates 

• 

• 

• 

Spot Testing 

Ferric chloride 

Mercuric chloride 

Meixner Test

Spectrochemical 

• Sophisticated spot tests 

• Chemical reaction to form lightabsorbing 

substance 

• Carefully controlled 

• Spectrophotometer vs eyeball

Co-oximetry 

• Spectrophotometry used to measure 

various forms of hemoglobin 

• Measurement of light absorbance at 

multiple wavelengths allows allows several 

hemoglobin species to be quantified 

• Need more wavelengths than 

types of hemoglobin


• Older tests waited until complete conversion 

• Modern tests measure rate of conversion 

• Initial phase, rate is constant and 

proportional to the initial 

concentration of the analyte 

• Nonreacting substances that absorb light 

• Don’t affect results


• Produce light absorbing 

substances 

• Ex: High 

concentrations of 

lactate 

• Inhibit the assay reaction 

or that consume 

reagents 

• Ex: ascorbic acid in 

oxidation reactions 

Oh, a drug test. That’s a relief. 

I thought you were going to test 

my ethics.


• Improve Selectivity 

• Enzymes that can catalyze highly 

selective reactions 

• Assays for ethanol use alcohol dehydrogenase 

• Rate of change of NAD + to NADH 

• Rate of increase in light absorption is 

proportional to ethanol concentration

Immunoassays 

• Created due to need to measure a very 

low concentraiton of an analyte 

• Quick, inexpensive 

• Combination of 

• High affinity 

• High selectivity 

• 2 common types

Immunoassays 

• Non-competitive 

• Analyte is sandwiched 

b/w 2 antibodies 

• Difficult with small drugs 

• Competitive 

• Analyte from specimen 

competes for number of Ab 

binding sites with a labeled 

version of the analyte

Non-competitive 

“Sandwich” assay

Competitive 

“Inverse” assay

Competitive 

From Goldfranks 9th edition

Immunoassays 

• Nonisotopic immunoassays are common 

• Limited to those in high demand 

• Lots of effort, high development cost 

• Low production cost (after development) 

• Homogenous immunoassays 

• Measure differences in bound 

and free labels

Immunoassays 

• Old - radio immunoassays 

• New - spectrophotometric 

• Enzyme multiplied immunoassay technique 

(EMIT) 

• Fluorescence polarization immunoassay (FPIA) 

• Kinetic inhibition of microparticles in solution 

(KIMS) 

• Cloned enzyme donor immunoassay (CEDIA)

EMIT 

Goldfranks 9th edition

Magnetic Microparticle 


Chromatography 

Separation and Detection 

•Encompasses several related techniques where analyte specificity 

is achieved by physical separation 

•Partition analytes between a stationary and mobile phase 

•Stationary: very fine particles aranged in a thin layer 

•Mobile (moving): phase flows thru spaces between particles 

•After separation--need a detection phase 

•However, can have provisional identification based on their 

characteristic velocities, distance traveled, or time to traverse 

the chromatography column 

•Not specific

Rf = 

Rf 

migration distance of substance 

migration distance of solvent front 

• Retention time 

• Time required to traverse the column 

• Retardation (planar) or retention (column) 

• Separation based on polarity, affinity, 

solubility, etc. 

• Standard for each analyte

Thin Layer 


• Extracts dissolved in a solvent 

• Placed on thin layer of silica gel 

• Plates placed vertically in closed tank 

• Solvent drawn upwards thru the gel 

• Xenobiotics are drawn up the gel 

• Hydrophobic rapidly 

• Hydrophilic slowly

TLC 


Thin Layer 


• Fast, easy, inexpensive 

• Polar silica medium 

• Low sensitivity (~ 1000 ug/L) 

• Low specificity 

• Used in drug screens 

• Requires large amount of material 

• Done on urine, gastric aspirate

TLC Drawbacks 

• Multiple steps 

• Slow, labor intensive 

• Interpretation of spots 

• Difficult to quantitate

High-Performance 

Liquid Chromatography 

• Stationary phase packed in a column 

• Mobile phase pumped through under pressure 

• Allows better separation in less time 

• Identification by retention time in a column 

• Also use ultraviolet spectroscopy 

• Measuring light absorbance allows the amount 

of the xenobiotic to be determined

Reverse phase 

chromatography 

• Non-polar stationary phase and 

hydrophilic mobile phase 

• Polar out first, non-polar retained 

• Hydrophobic stationary 

• Hydrophilic mobile phase 

• Most common HPLC technique 

• TLC can be done this way too

High-Performance 

Liquid Chromatography 

• Expensive, complex, fast 

• Separation under pressure 

• Quantitation, detection > 10 ug/L 

(10 fold difference) 

• 

• 

• 

• 

Low specificity for same class 

One at a time; narrow range of polarity 

Measure serum concentration for which no 

immunoassay is available 

Can’t analyze multiple specimens

HPLC

Gas Chromatography 

• Similar to HPLC except the moving phase is a gas 

• Nitrogen, helium 

• Low flow resistance of gas 

• High flow rates 

• Less time 

• Temperature gradient allows multiple xenobiotics 

to be analyzed at once 

• Partitioning depends on natural volatility 

• Temp < 572° F (300° C )


• Expensive 

• Screening or confirmatory 

• High specificity and sensitivity 

• Quantitation and broad screening 

• Temperature gradient 

• Analyte volatilized in injector, column kept hot 

• Heat labile cannot be assessed


• Substances have characteristic retention 

times 

• Column outflow detectors 

• Flame-ionization - organic compounds 

• Most common detector 

• Nitrogen-phosphorous - N or P 

compounds (many drugs)

Mass Spectrometry 

• Can serve as highly sensitive GC detector 

• Analyte separated from the gas carrier and 

then filtered to a detector 

• Extremely high specificity 

• Unparalleled ID of organic chemicals 

• Shoots electrons → fragment ions 

• Characteristic of the parent molecule

Mass Spectrometry

MS Limits 

• Cannot separate enantiomers 

• D-methamphetamine (DOA) 

• L-methamphetamine (inhalers) 

• Vick’s inhalers, metabolite of selegiline 

• “L” = legal 

• Can separate by a chiral analysis

LC/MS/MS 

• High sensitivity and specificity extended by 

the related hybrid technique of liquid 

chromatography/tandem mass 

spectrometry

GC/MS 

• Expensive equipment 

• Most common gold standard, in ~1 hour 

• Most specific and sensitive 

• Sensitivity 2 – 10 ug/L

Relative Comparison 

Method Speed Cost 

Spot Test Fast $ 

Spectrochemical Medium $ 

Immunoassay Medium $$ 

TLC Slow $$ 

HPLC Medium $$ 

GC Medium $$ 

GC/MS Slow $$$ 

LC/MS/MS Medium $$$$ 

*Reproduced from Goldfranks 9th edition

Hair Analysis 

• Hair grows ~1 cm/month 

• Can utilize multiple methodologies 

• Contamination is greatest flaw

Drug Screens

• Amphetamines 

• Cannabinoids 

• Cocaine 

• Opiates 

• Phencyclidine 

NIDA-5

• 

Highly specific 

• Benzoylecgonine 

• 

• 

• 

• 

Inactive metabolite 

No false positives 

Acute: 2-3 days 

Chronic: 1 week 

Cocaine

• 

• 

• 

• 

• 

Tetrahydrocannabinol 

Tetrahydrocannabinoic acid 

Inactive metabolite 

Occassional use: 3 days 

Chronic use: > 1 month 

11-OH THC

• 

• 

• 

• 

• 

• 

The “second hand 

smoking” defense 

Studies of passive 

exposure 

Confined areas 

Mixed results 

Cutoffs: 20 ng/ml 

Levels: ~6 ng/ml 

NIDA 50 ng/ml

• Dronabinol 

• Efavirenz 

• NSAIDs 

• Promethazine 

• Riboflavin 

• 

Ethacryinc acid 

THC 

False Positives

• 

• 

• 

• 

Morphine, codeine, 

heroin 

Cross reactivity 

depends on assay 

Synthetics show 

little or no 

cross-reactivity 

Specific directed 

assays available 

Opioids

“Poppy Seed” Defense 

6-MAM

Challenges 

Dextromethorphan 

• Dextrorphan – major metabolite 

• Levorphanol – “L” enantiomer, also opioid 

• Can’t differentiate optical enantiomers by MS

Methadone 

False Positives 

• Quetiapine 

• Doxylamine 

• Olanzapine 

• Diphenhydramine & 

Verapamil metabolites 

*Tests for other sythetic and semisynthetics are available

• Phencyclidine 

• 

• 

• 

False Positives 

DXM, Ketamine 

• Diphenhydramine 

• 

• 

Venlafaxine, bupropion 

Metabolites of PCP 

False Negatives? 

PCP

• 

Benzodiazepines 

Metabolized to 

oxazepam 

• Chlordiazepoxide 

• Diazepam 

• Temazepam 

• Glucuronides 

(lorazepam)

Amphetamines 

• 

• 

• 

Amphetamine assay plagued 

with false positives 

Fails to detect “designer” 

amphetamines 

Bupropioin (cathinone) 

• Pseudoephedrine

Nasal Inhalers 

• Can contain l-methamphetamine 

• less potent isomer of d-methamphetamine 

• Both turn immunoassays positive 

• Difficult to distinguish with mass spec 

• Optical enantiomers

TCA Challenges 

• Cross react with ringed xenobiotics 

• Carbamazepine 

• Phenothiazines 

• Diphenhydramine 

• Major challenge is timing

Federal Cutoffs (ng/mL) 

Substance Screening Confirmatory 

Cocaine (met) 300 ng/ml 150 ng/ml 

Opiate (met) 2000 ng/ml 2000 ng/ml 

Amphetamines 1000 ng/ml 500 ng/ml 

THC (met) 50ng/ml; 1000 ng/ml THCA 15 ng/ml THCA 

PCP 25 ng/ml 25 ng/ml

Adulteration 

• 2004: First mandatory guidelines for federal 

workplace testing (SAMHSA) 

• Tampering 

• In vivo adulteration 

• In vitro adulteration 

• Urine substitution 

• Little evidence to support any 

product working consistently

Specimen Validity 

• Appearance 

• Temperature 

• 90-100 o F 

• pH testing 

• pH 3-11 

• Specific gravity 

• Cr < 20 mg/dl 

• > 1.003 

• Creatinine 

• > 20 ppm

• 

• 

• 

• 

• 

• 

In Vivo Adulteration 

“Ingest prior to urination” 

Primary mechanisms: dilution and excretion 

Water and diuretics 

Fool visual inspection 

Interfere with creatinine level checks 

Water, Naturally Klean herbal tea, 

golden seal, HCTZ 

B-vitamins, riboflavin, creatinine 

• 

Riboflavin may cause fluorescent urine

In Vitro Adulteration 

“Add after urination” 

• Interfere with an immunoassay or convert a 

target drug to a different compound 

• Sold under many names but contain: 

• Glutaraldehyde 

• Sodium or potassium nitrate 

• Pyridinium chlorochromate 

• Peroxide/peroxidase 

• Household products, too

• 

• 

• 

• 

• 

• 

In Vitro Adulteration 

“Add after urination” 

Glutaraldehyde: interferes with immunoassay, denatures 

Nitrite: decreased ion concentration 

Pyridinium: decrease in pH levels 

Peroxidase: oxidizing drugs and metabolites 

Bleach: decreases detection 

Vinegar: interferes with detection; lowers pH

Others 

• Acids and bases (i.e. lemon juice, NaOH) 

• Oxidizing agents (i.e. bleach, peroxide) 

• Denaturants (i.e. glutaraldehyde) 

• Eyedrops (i.e. benzalkonium chloride) 

• Reduces binding of immunoassay 

• Soap (created FP and FN)

Urine Substitution 

Ontario Smith

Questions?

Cholinesterases 

• Butyrylcholinesterase (plasma cholinesterase) 

• Metabolizes cocaine, succinylcholine 

• Falls first, recovers first 

• Red Cell Acetylcholinesterase 

• Reflects activity at the NMJ 

• Low concentrations in people 

• Serve as markers for poisonings

Butrylcholinesterase 

• Malnutrition 

• 

• 

• 

• 

• 

Cholinesterases 

Hereditary deficiency 

Iron deficiency anemia 

Hepatic disease 

Chronic Illness 

Succ, codeine, morpine 

• 

Pernicious anemia 

• Antidepressants 

• Antimalarials 

• Hemoglobinopathies 

• 

Red Cell 

Inc with oral contraceptives

Anion Gap Reliability 

AG = Na + - (Cl - + HCO3 - ) 

• MUDPILES 

• Cyanide, CO, Acetaminophen, Toluene, 

Theophylline, Hydrogen Sulfide 

• Increase unmeasured anions 

• Dehydration, sodium salts, antibiotics 

• Decreased unmeasured cations 

• Mag, Ca, and K

Anion Gap Reliability 

• Hypercalcemia 

• Hypermagnesemia 

• Hyperkalemia 

• Lithium 

• Multiple myeloma 

Low Anion Gap 

• Hypoalbuminemia 

• Bromism 

• Iodism 

• Nitrate excess

Forensic Toxicology

• 

• 

• 

Forensics 

Aid medical / legal investigation of death, 

poisoning, and drug use 

Concern is not legal outcome, but obtaining 

and interpreting results 

Chain of Custody 

• 

• 

List everyone who handled a specimen 

(special couriers) 

Where specimen was at any given time

DEA Schedule 

• Controlled Substances Act (1970) 

• I - High abuse potential, no medical use 

• Heroin, PCP, LSD, GHB, GHB MDMA, etc. 

• II - High abuse potential, but has medical use 

• Most opioids, barbiturates, methylphenidate, etc. 

• No refills 

• III - Ketamine, buprenorphine, GHB 

• IV - BZD, long acting barbiturates, modafinil 

• V - Codeine cough suppressants, pregabalin, diphenoxylate

Medical Review Officer 

• 

• 

Licensed physician 

“Expert in drug and alcohol testing and the 

application of federal regulations to the process.” 

• Consultant 

• Business, industry, labor, government or 

academia 

• 

Relating to prevention, detection and control 

of drug abuse in the workplace

Drug Abuse Testing 

• Strict, invariable procedures, federally mandated (DOT) 

• Certified lab, separate from all other testing 

• MRO interpretation required 

• COAT-PE 

• ‘SAMHSA 5’ - cocaine, opioids, amphetamine, THC, 

PCP 

• ETOH breath testing 

• Screening, then confirmatory if + by cutoffs

Post Mortem 

Toxicology

Post Mortem Changes 

“Necrokinetics” 

• Postmortem interval 

• Defined by the degree of decomposition 

• Decomposition 

• Autolysis: enzymes are released and 

chemicals move down gradients 

• Putrefacation: digestion by bacteria 

• Anthropophagia: feeding on remains

Specimens 

• Blood 

• Reported as “blood concentrations” 

• From femoral or subclavian (low glucose) 

• Right heart blood (elevated glucose) 

• Vitreous 

• Avascular, acellular so well 

protected 

• Aqueous content > blood 

• Urine 

• Bladder serves as a “reservoir”

Interpretation Confounders 

• Postmortem redistribution 

• Postmortem metabolism 

• Continuous absorption 

• Xenobiotic stability 

• Chemical interactions 

• Expected clinical effects 

• Comorbid, tolerance, genetics

Other sources

Redistribution Doesn’t Occur 

Leikin JB. Clin Tox 2003;41(1):47-56.

Legal Ethanol

Legal Ethanol 

• State determines own legal driving limit 

• DWI, DUI, DWAI, etc. 

• Zero tolerance 

• “Illegal per se” 

• What is “drunk”? 

• ~ 150 mg/dL

The Barman’s Paradox 

• Legally drunk 50-80 mg/dL or 

0.05-0.08 g/dL [%] 

• Serving to intoxicated is prohibited 

• No better at determining “drunk”

Blood Ethanol Testing 

• The law = whole blood 

• The lab = serum / plasma 

• [serum] = [plasma] 

• ETOH does not enter RBCs well 

• [serum]/[blood] ~ 1.15 

• The lab measures higher than the law 

• [Breath ethanol] mmol/L X 2100 = [Blood ethanol] mmol/L

1. Wide turns 

2. Straddling / driving on 

lane marker 

3. Nearly striking object or 

another vehicle 

4. Weaving / swerving 

5. Going too slow (> 10 

MPH below speed limit) 

The Clues 

6. Stopping inappropriately / 

without cause 

7. Following too closely 

8. Erratic braking 

9. Driving into opposing / 

crossing traffic 

10.Slow response to traffic 

signals 

*NHTSA Top 10 in descending order of probability of intoxication


What’s in a Drink? 

• 10 oz beer (5%) 

• 4 oz of wine (12%) 

• 1 oz liquor (50%) 

• All ~ 15 g ETOH 

• 100-125 mg/kg/hr 

• Avg adult: 7-10 g/hr 

or 15-20 mg/dL/hr 

• Tolerance: 30 mg/dL/hr 

Approximately

Laboratory Methods 

• Enzymatic 

• ADH + ETOH = NADH (340 nm) 

• FP with elevated lactate (lactate 

converted to pyruvate increases 

NADH formation) 

• GC 

• Can detect other volatiles (“toxic”)

• Specific gravity 0.8 g/ml 

• Vd 0.6 L/kg 

• 1-1.5 shot(s) = 30 ml 

• mmol/L = (mg/dL)/4.6 

• % = grams/100 ml 

The Numbers

Sample Calculation 

50 kg man drinks 1 shot of 80 proof alcohol 

1.5 oz (30 ml/oz) = 45 ml 40% of 45 ml = 18 ml 

18 ml X 0.8 g/ml = 16 g which is 16,000 mg 

50 kg (0.6 L/kg) = 30 L (10 dL /L) = 300 dL 

16,000 mg/300 dL = 80 mg/dL

• 

• 

• 

• 

• 

• 

“Breathanol” 

[expired air] surragote for [blood] (sort of) 

• 

Henry’s law 

[ethanol] remains constant throughout respiratory 

tract 

Mean breath:blood ratio 1:2100 used in forensics 

Many confounders 

[Breath ethanol] mmol/L X 2100 = [Blood ethanol] mmol/L 

Breath units underestimate BAC

1. Goldfranks 9th Edition 

References 

2. Jaffee WB et al. Is this urine really negative? A 

systematic review of tampering methods in UDS 

and testing. J of Subs Abu Treat 2007;33:33-42. 

3. Leikin JB, et al. Post-mortem toxicology: What the 

dead can and cannot tell us. Clin Tox 2003;41(1): 

47-56. 

4. Shannon: Haddad and Winchester’s Clinical 

Management of Poisoning and Drug Overdose, 4th 

edition 2007

Photo References 

1. http://www.offthemark.com/cartoons/1998-12-22.gif 

2. http://www.ecardica.com/ecards/postcards%5Cfunny%20pictures/drug_testing_at_pepsi.jpg 

3. http://www.flickr.com/photos/cornellfungi/2828851149/sizes/z/in/photostream/ 

4. http://www.cartoonstock.com/newscartoons/cartoonists/jlv/lowres/jlvn386l.jpg 

5. http://cdn1.images.videobash.com/photos/000/022/977/22977.jpg 

6. http://26.media.tumblr.com/tumblr_lykxob8LmS1qfcutbo1_500.jpg 

7. http://www.thcfinder.com/uploads/files/marijuana-mcdonalds.jpg 

8. http://www.cannabisculture.com/v2/files/images/2493-homer-phish.jpg 

9. http://www.myfreewallpapers.net/movies/pages/the-good-the-bad-and-the-ugly.shtml 

10. http://www.sodahead.com/entertainment/bristol-palin-gets-her-own-reality-show-laughable-or-legit/question-1778221/?page=4&link=ibaf&q=meth 

+mouth&imgurl=http://farm4.static.flickr.com/3063/2780829321_9f3f10177b.jpg 

11. http://images.teamsugar.com/files/upl0/1/13254/08_2008/headline.preview.jpg 

12. http://www.cdc.gov/Features/AlcoholConsumption/ 

13. http://sharetv.org/shows/seinfeld/episodes/346227 

14. http://ourweed.com/funny-marijuana-pictures/ 

15. http://cdn.bleacherreport.net/images_root/slides/photos/000/840/267/52000365_display_image.jpg?1302153389 

16. http://www.4aceswholesale.com/cart/images/New-Whizzinator-w.jpg 

17. http://theestreet.com/wp-content/uploads/2012/01/entourage.jpg 

18. http://thebeautybrains.com/2007/08/16/the-scandalous-secret-beauty-companies-dont-want-you-to-know/ 

19. http://2pep.com/funny%20pics/cool_funniest_hilarious_pictures/ 

super_funny_hilarious_pictures_of_25_epic_drunk_shaming_photos_20090603_1483976130.jpg 

20. http://www.cartoonstock.com/newscartoons/cartoonists/mba/lowres/mban1080l.jpg 

21. http://www.thecrimsoncrow.com/wp-content/uploads/2011/05/heroin-up-his-butt-billboard-12835-1306154078-11.jpg 

22. http://images3.wikia.nocookie.net/__cb20111221073446/simpsons/images/7/7b/Eliza_Simpson_Detective.png 

23. http://amazingdata.com/mediadata57/Image/crazy_funny_amazing_shocking_awesome_sexy_200907311639075608.jpg 

24. http://mimg.ugo.com/201103/3/8/7/176783/cuts/charlie-sheen-crazy-eyes_288x288.png

Analytical and Forensic Toxicology

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